In the field of PDT, various tetrapyrrolic macrocycles, such as purpurins, chlorins, bacteriochlorins, phthalocyanines and benzochlorins have shown the ability both to localize at a tumor site and to absorb light to form an activated state in response to the light. These macrocycles then exhibit a cytotoxic effect on the cells or other tissues in which they are localized when irradiated at the appropriate wavelength.
To cause the desired phototoxic effect deep within a subject's tissue, however, it is necessary to use photosensitizers that possess high absorption coefficients at wavelengths longer than 650 nm, where body tissues are most transparent to light. See Sternberg et al., "An Overview of Second Generation Drugs for Photodynamic Therapy Including BPD-MA (Benzoporphyrin Derivative)", Photodynamic Therapy and Biomedical Lasers, 470-4 (Spinelli et al. eds. 1992).
The reduction of a porphyrin to form a chlorin (i.e., a dihydroporphyrin) changes the optical properties in this desirable way, and reducing the chlorin further to form a bacteriochlorin (i.e., a tetrahydroporphyrin) makes the desired effect even more pronounced. There has only been one general method known to convert meso-tetraphenyl porphyrins into the corresponding chlorins, namely the diimide reduction introduced by Whitlock et al., "Diimide Reduction of Porphyrins", J. Am. Chem. Soc., 91, 7485-89 (1969). However, the product produced does not have a .beta.,.beta.'-dihydroxy substitution pattern.
In addition to the desirable absorptive properties of chlorins and bacteriochlorins, the amphiphilic character of these compounds has been pointed out as being potentially beneficial with respect to the desired biodistribution of the drug. For example, Bonnett et al., "Second Generation Tumour Photosensitisers: The Synthesis and Biological Activity of Octaalkyl Chlorins and Bacteriochlorins with Graded Amphiphilic Character", J. Chem. Soc., Perkin Trans. 1, 1465-70 (1992), have suggested that meso-tetra(hydroxyphenyl)chlorins and their corresponding bacteriochlorins could be used as photosensitizers in PDT.
It is known that .beta.-substituted porphyrins can be treated with osmium tetroxide (OsO.sub.4) to oxidize one or more double bonds, thus forming an osmate ester at the .beta.,.beta.'-position, which can then be reduced with any one of a variety of reducing agents to form the corresponding vicinal-diol. For example, in Chang et al., "A Novel Method of Functionalizing the Ethyl Chain of Octaethylporphyrin", J. Org. Chem., 52, 926-29, the corresponding diol was obtained by oxidizing octaethylporphyrin with OsO.sub.4 in the presence of pyridine. ##STR2## Osmylation of a completely .beta.,.beta.'-alkyl substituted, 5,15-bis-(methylphenyl)porphyrin has similarly produced the corresponding diol. Osuka et al., "Synthesis of 5,15-Diaryl-Substituted Oxochlorins from 5,15-Diaryl-octaethylporphyrin", Bull. Chem. Soc. Jap., 66, 3837-39 (1993).
However, the diols so produced tend to undergo a pinacol-pinacolone-type rearrangement when exposed to acidic conditions, yielding oxochlorins, as shown below: ##STR3## When the migratory aptitude of various substituents was studied, it was established that, from the rearrangement of the .beta.-monoalkyl-substituted diols, hydrogen was the "substituent" with the greatest tendency to migrate in a rearrangement reaction. Chang et al., "Migratory Aptitudes in Pinacol Rearrangement of vic-Dihydroxychlorins", J. Heterocyclic Chem., 22, 1739-41 (1985).
Vicinal-dihydroxychlorins have been obtained from .beta.,.beta.'-alkyl substituted porphyrins by oxidation with osmium tetroxide in pyridine, and it has been confirmed that the product undergoes a pinacol rearrangement on treatment with sulfuric acid. See Bonnett et al., "The Oxidation of Porphyrins with Hydrogen Peroxide in Sulphuric Acid", Proc. Chem. Soc., 371-72 (1964), and Chang et al., "Differentiation of Bacteriochlorin and Isobacteriochlorin Formation by Metallation. High Yield Synthesis of Porphyrindiones via OsO.sub.4 Oxidation", J. Chem. Soc., Chem. Commun., 1213-15 (1986). However, it has not been thought that the dihydroxy osmylation product of a .beta.,.beta.'-unsubstituted, meso-substituted porphyrin would be stable in view of the likelihood of rearrangement.
Further, if the starting porphyrin bears a .beta.-substitution pattern, which lowers the overall symmetry of the molecule, dihydroxylation leads to a non-statistical mixture of stereo- and regioisomers. For example, when the dimethyl ester of deuteroporphyrin-IX is osmylated, a mixture of the following regioisomers and their corresponding stereoisomers is produced. Chang et al., "C-Hydroxy- and C-Methylchlorins. A Convenient Route to Heme d and Bonellin Model Compounds", J. Org. Chem., 50, 4989-91 (1985). ##STR4## Under the best of conditions, the separation of these regioisomers and stereoisomers is cumbersome.
It has now been found that .beta.,.beta.'-unsubstituted, meso-substituted porphyrin compounds can be .beta.,.beta.'-dihydroxylated via the addition of OsO.sub.4, followed by reduction to give the vic-diol, as shown below: ##STR5## The resulting meso-substituted vic-diols are unexpectedly stable. Surprisingly, dehydration and rearrangement only takes place under relatively harsh conditions, such as treatment with refluxing benzene containing catalytic amounts of HClO.sub.4. This is unexpected in view of, not only the high migratory aptitude of the .beta.-hydrogens, but also the expected tendency of the molecule to eliminate water, thus reconstituting a fully conjugated, porphyrin resonance structure as the enolic tautomer, as shown below. Crossley et al., "Tautomerism in 2-Hydroxy-5,10,15,20-tetraphenylporphyrin: An Equilibrium Between Enol, Keto, and Aromatic Hydroxyl Tautomers", J. Org. Chem., 53, 1132-37 (1988). ##STR6## Such meso-phenyl oxoporphyrins have been previously prepared via a fundamentally different route. See, e.g., Catalano et al., "Efficient Synthesis of 2-Oxy-5,10,15,20-tetraphenylporphyrins from a nitroporphyrin by a Novel Multi-Step Cine-substitution Sequence", J. Chem. Soc., Chem. Comm., 1537-38 (1984).
It has been found that, when hydroxy groups are added to a pre-existing meso-substituent, for example, the phenyl substituents in meso-tetra(hydroxyphenyl)-porphyrins, chlorins and bacteriochlorins can be effective as active PDT agents. See Berenbaum et al., "Meso-Tetra(hydroxyphenyl)-porphyrins, a New Class of Potent Tumour Photosensitisers with Favourable Selectivity," Br. J. Cancer, 54, 717-25 (1986) and Ris et al., "Photodynamic Therapy with m-Tetrahydroxyphenylchlorin in vivo: Optimization of the Therapeutic Index", Int. J. Cancer, 55, 245-49 (1993). By introducing hydroxy-functionalities into the .beta.-positions, not only has a new class of photosensitizer compounds been found, but there is reason to believe that the photosensitizers of the invention are even superior to known compounds due to enhancement of amphiphilicity of the molecule.
Further, upon .beta.,.beta.'-dihydroxylation, the high symmetry of the starting materials causes the formation of only one regio- and stereoisomer of the resulting chlorin. For example, the dihydroxylation of meso-tetraphenylporphyrin generates only one isomer of .beta.,.beta.'-dihydroxy-meso-tetraphenylbacteriochlorin. Further still, subsequent .beta.,.beta.'-dihydroxylation of the .beta.,.beta.'-hydroxychlorin generates only two, easily separable diastereomers of the tetrahydroxybacteriochlorin product. This significant reduction of isomers provides a method for obtaining PDT agents in high yields, which is of great practical, economical and medicinal importance.
Consistent with previous observations (see, e.g., Whitlock et al , "Diimide Reduction of Porphyrins" J. Am. Chem. Soc., 91, 7485-89 (1969) and Chang et al. "Differentiation of Bacteriochlorin and Isobacteriochlorin Formation by Metallation: High Yield Synthesis of Porphyrindiones via OsO.sub.4 Oxidation", J. Chem. Soc., Chem. Comm., 1213-15 (1986)), the .beta.-hydroxylation of .beta.,.beta.'-dihydroxychlorins (and the diimide reduction .beta.,.beta.'-dihydroxychlorins or, for that matter, the .beta.,.beta.'-dihydroxylation of tetraphenylchlorins) are susceptible to a pronounced metal-directing effect. Osmylation/reduction of metallochlorins produces a metallo-isobacteriochlorin chromophore, from which the parent isobacteriochlorin chromophore can be obtained by demetallation. In contrast, osmylation/reduction of the free base chlorins produces the corresponding bacteriochlorin chromophores.
Yet another advantage is that the meso-substituent can be widely derivatized, particularly when it is an aryl ring, such as a phenyl group. Thus, by hydroxylating .beta.,.beta.'-unsubstituted, meso-substituted porphyrins and chlorins via oxidation with OsO.sub.4, followed by reduction of the intermediate osmate ester formed at the .beta.,.beta.'-position, there can be made a number of related vic-diol substituted chlorins and bacteriochlorins exhibiting particularly desirable characteristics as PDT agents, such as intensified and bathochromically shifted Q bands and increased amphiphilicity. Moreover, due to the ability to further derivatize the meso-substituents themselves, an opportunity is provided for fine-tuning the pharmacokinetics and -dynamics of the compounds to an even greater degree.